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Premium member Presentation Transcript Screening of drugs used in parkinsonism: Screening of drugs used in parkinsonism Presented by : YASHWANTH B.MParkinsonism : Parkinsonism INTRODUCTION This disease was first studied & discovered by James Parkinson (1817) in his Essay on the Shaking Palsy. The pathology of PD was not well understood until the early 20th century, when the German pathologist Frederick Lewy in 1912 reported neuronal cytoplasmic inclusions in a variety of brain regions. In 1919, Tretiakoff observed that the most critical abnormality in PD was the loss of neurons in the substantia nigra pars compacta of the midbrain. In the 1950s, investigators discovered the importance of dopamine and its depletion from the basal ganglia as the key to understanding the pathophysiology and pathologic biochemistry of PD. There is some evidence that a disease known as " kampavata ", consisting of shaking ( kampa ) and lack of muscular movement ( vata ), existed in ancient Indian medical system, Ayurveda , as long as 4500 years ago .The Mucuna pruriens plant was used in ancient times to treat the symptoms, and was later discovered to contain levodopa . 2PowerPoint Presentation: DOPAMINE: it is a principle inhibitory NT in the brain whose distribution is restricted to the dopaminergic neurons of substantia nigra (extra pyramidal motor center), limbic system & hypothalamus. Lie all catecholamines most of dopamine secreted in the synapse is recaptured by reuptake mechanism involving dopamine transporters. They are also degraded by MAO & COMT. ACETYLCHOLINE : it is an excitatory NT which is well distributed in various regions in the brain & also secreted by the cholinergic neurons of the caudate nucleus in the basal ganglia. Along with dopamine they together regulate the motor function. Ach is quickly degraded in the synapse by choline esterase enzyme. 3PowerPoint Presentation: DEFINITION: Parkinson ’ s disease is a chronic progressive degenerative disorder of the CNS caused by an imbalance b/w dopaminergic & cholinergic activity in the brain due to degeneration of dopaminergic neurons resulting in depleted levels of dopamine in the brain leading to motor function disorder & various other extra pyramidal symptoms as follows. Cardinal features of Parkinson ’ s disease: 1) Rigidity: due to increased muscle tone. 2) Tremors : colinergic overactivity . 3) Akinesia / hypokinesia : slowness in the movement. 4Other extra pyramidal symptoms of Parkinsonism: : Other extra pyramidal symptoms of Parkinsonism: 1) Silarrohea –excessive salivation from the mouth. 2) microphagia - small handwriting. 3) Pill rolling -tremors at 4-8 cps (resting). 4) Mask like facial expression 5) Typical gait -no swinging of hands 6) Defective posture -stooped/hunched back. 7) Excessive blinking & dementia (impaired memory) 8) Decreased speech volume. 5BASAL GANGLIA: BASAL GANGLIA 6 Basal nigrostriatal tract substantia ganglia nigra Basal ganglia: consists of three nuclei (grey matter cell bodies) i.e. - globus pallidus , & corpus striatum ( putamen & caudate nucleus). Corpus striatum communicates with substantia nigra via dopaminergic neurons (dopamine NT) that constitutes the nigrostriatal tract. Over time Substrantia nigra undergoes atrophy as dopaminergic neurons continue to diminish & get degraded in Parkinsonism.PowerPoint Presentation: 7PowerPoint Presentation: 8PowerPoint Presentation: 9ETIOLOGY AND PATHOGENESIS OF PARKINSON’S DISEASE : ETIOLOGY AND PATHOGENESIS OF PARKINSON’S DISEASE ETIOLOGY The specific etiology of Parkinson’s disease (PD) is not known. Epidemiologic Studies indicate that a number of factors may increase the risk of developing Parkinsonism. Genetic & age related Factors Approximately 5–10% of PD patients have a familial form of Parkinsonism with an autosomal -dominant pattern of Inheritance. Large pedigrees have been identified where members in different generations suffer from PD. Parkinsonism is also typically observed in older subjects around the age of 60 & above. 10PowerPoint Presentation: Environmental Factors The most compelling evidence for an environmental factor in PD relates to the toxin 1, 2, 3, 6-methyl-phenyl-tetrahydropyridine (MPTP). MPTP is a byproduct of the illicit manufacture of a synthetic meperidine derivative (used as an adulterant to heroin ) Drug addicts who took MPTP developed a syndrome that strikingly resembled PD, both clinically and pathologically MPTP induces toxicity through its conversion in astrocytes to the pyridinium ion (MPP C ) in a reaction catalyzed by monooxidase type B ( MAO-B ). MPP C is then taken up by dopamine neurons and causes a mitochondrial complex I (ETC) defect similar to that found in PD .This observation supports the possibility that an environmental factor might cause PD; however, no MPTP-like factor has been identified in PD patients to date 11PATHOGENESIS : PATHOGENESIS Oxidative Stress:- Because of the potential of the oxidative metabolism of dopamine to yield hydrogen peroxide (H2O2) and other reactive oxygen species ( ROS ).Oxidant stress and consequent cell death could develop in the SNc under circumstances in which there is ( a ) increased dopamine turnover, resulting in excess peroxide formation; ( b ) a deficiency in glutathione (GSH), thereby diminishing the brain’s capacity to clear H2O2; or ( c ) an increase in reactive iron, which can promote OH radical formation. Indeed, postmortem studies in PD brains demonstrate increased iron, decreased GSH, and oxidative damage to lipids, proteins, and DNA, suggesting that the SNc is in a state of oxidant stress. 12IRON : IRON Numerous studies, using a variety of analytical techniques, have demonstrated that iron levels are increased within the substantia nigra of PD patients. Infusion of iron into the SNc of rodents induces a model of PD characterized by a concentration-dependent and progressive loss of striatal dopamine. degeneration of SNc neurons , behavioral changes and further increase in SNc iron has been observed following MPTP treatment or 6-hydroxydopamine (6-OHDA). 13PowerPoint Presentation: Both the enzymatic and the chemical metabolism of dopamine result in the formation of Hydrogen peroxide (H2O2) ( a and b ). H2O2 is normally cleared by reduced glutathione (GSH) ( c ). However, an increase in the steady-state concentration of H2O2 can lead to a reaction with ferrous iron that generates the highly reactive and potentially cytotoxic hydroxyl radical (OH) which degrades dopaminergic neurons. 14GLUTATHIONE : GLUTATHIONE A defect in one or more of the naturally occurring antioxidant defenses could prevent neurodegeneration in PD. A reduction in GSH may impair H2O2 clearance and promote OHq formation, particularly in the presence of increased iron. The cause of the decrease in GSH in PD is unknown. A defect in GSH, comparable to that found in PD, has been detected in the nigra of patients discovered at autopsy to have incidental Lewy bodies (ILB). buthionine sulphoximine (BSO), a selective inhibitor of ®- glutamylcysteine synthetase , induces a reduction in GSH and is toxic to cultured dopaminergic neurons. In rats, administration of BSO sufficient to induce a 40–60% decline in GSH, paralleling the degree of loss that occurs in PD, does not affect the number of tyrosine hydroxylase (TH)–positive cells in the SNc However, this level of GSH depletion enhances the neurodegeneration that is observed when the rodents are treated with other toxins such as 6-OHDA .These observations suggest that a reduction in GSH by itself may not damage dopamine neurons but may render them vulnerable to other toxins. 15PowerPoint Presentation: OXIDATIVE DAMAGE There is evidence of oxidative damage in the brains of PD patients. Increased levels of the lipid peroxidation products malondialdehyde (MDA) and lipid hydroperoxide have been found in the SNc . However, the majority of PD patients receive levodopa therapy, and it is uncertain if its oxidative metabolites contribute to the oxidative damage detected postmortem. Levodopa has been shown to induce degeneration of cultured dopamine neurons but has not been shown to be toxic to dopamine neurons in normal rodents and humans. The situation may be different in PD where defense mechanisms are impaired. Indeed, levodopa can augment neuronal degeneration and increase lipid peroxidation in rodents pretreated with 6-hydroxydopamine. 16PowerPoint Presentation: Other mechanisms : Mitochondrial Dysfunction - ETC blockade . Excitotoxicity Neurotrophic Factors Glia Immune Modulators 17Treatment of Parkinsonism : Treatment of Parkinsonism ( A)Drugs acting on dopaminergic system: (1) Dopamine precursors : L-dopa- (2) Dopa decarboxylase inhibitors: carbidopa & benzarazide (3) Dopamine agonists: bromocryptine , pergolide , peribidil , ropinorole , pramipexole (4) Dopamine facilitators: amantidine (5) MAO-B inhibitor: seliginine (6) COMT-inhibitors: entacapone , tolacapone 18PowerPoint Presentation: (B) Drugs acting on central cholinergic system: Centralanticholinergics : trihexiphenidyl ’ procyclidin,biperidin . (2) Central antihistaminics ; orphenadrine,promethazine - also exhibit anticholinergic action. 19Screening models for antiparkinson drugs :: Screening models for antiparkinson drugs : INVIVO MODELS : Tremorine And Oxotremorine Antagonism Mptp Model In Monkeys Resesrpine Antagonism Circling Behavior In Nigostriatal Lesioned Rats Elevated Body Swing Method Skilled Paw Reaching In Rats Stepping test in rats 20Screening models for antiparkinson drugs :: Screening models for antiparkinson drugs : INVITRO METHODS: 1.Experiment using rat striatal slices 2.Dopamine stimulated adenyl cyclase activity 3.Radioligand binding studies for D1 & D2 dopamine receptors 4.Dopamine release from synaptosome . 5. In vitro neuroprotective efficacy. 21In-vivo models: In-vivo models 22TREMORINE AND OXOTREMORINE ANTAGONISM:: TREMORINE AND OXOTREMORINE ANTAGONISM: PURPOSE AND RATIONALE The muscarinic agonists tremorine and oxotremorine induce parkinsonism-like signs such as tremor, ataxia, spasticity, salivation, lacrimation and hypothermia. These signs are antagonized by central anticholinergic drugs. 23PowerPoint Presentation: 24 PROCEDURE: Group of 6-10 male NMRI mice (18-22g) Dosed orally with test/ standard (5mg/kg benzatropine mesilate ) 1h prior administration of 0.5mg/kg oxotremorine (S.C) Before administration of oxotremorine (S.C) rectal temperature is measured & taken as basal value. Rectal temperature is again measured after 1,2,3h after oxotremorine inj. Tremor is scored after oxoremorine dosage at10sec observation duration for every 15min interval for 1h and scored.PowerPoint Presentation: 25 Salivation and Lacrimation are also scored every15min and 30min after oxotremorine inj. SCORES AS FOLLOWS: TREMOR, SALIVATION & LACRIMATION SCORE Absent 0 Slight 1 Medium 2 Severe 3 Test drug & standard should reduce the scorePowerPoint Presentation: 26 EVALUATION Hypothermia The differences of body temperature after 1, 2 and 3 h versus basal values are summarized for each animal in the control group and the test groups. The average values are compared statistically. Tremor The scores for all animals in each group at the 3 observation periods are summarized . The scores in the treated groups are expressed as percentage of the of the scores in control group. Salivation and lacrimation The scores for both symptoms for all animals in each group are summarized at the 2 observation periods. The numbers in the treated groups are expressed as percentage of the number of the control group.PowerPoint Presentation: CRITICAL ASSESSMENT OF THE METHOD The oxotremorine antagonism has been proven to be a reliable method for testing central anticholinergic activity. The overt isomorphism between the animal model and the symptoms of Parkinson’s disease recommend this test for screening of anti-Parkinson drugs. However, the model measures only central anticholinergic activity & cannot be used to screen dopaminergic drugs. 27Reserpene antagonism : Reserpene antagonism PURPOSE AND RATIONALE Reserpine induces depletion of central catecholamine stores. The sedative effect can be observed in mice shortly after injection, followed by signs of eyelid ptosis , hypokinesia, rigidity, catatonia, and immobility. These phenomena can be antagonized by dopamine agonists. 28PowerPoint Presentation: 29 PROCEDURE: Male NMRI mice (20-25g) used. Inject reserpine I.P (5mg/kg) & tested after 24h 30min prior to observation test compounds are injected(23.30 hrs). NMRI mice are Placed singly onto the floor of a Perspex container of dimentions (30× 26 cm, 20 cm) high,situated on a panlab proximity sensor Horizontal movements are recorded for 10min. (rearing and grooming episodes are also registered by an experienced observers.PowerPoint Presentation: 30 EVALUATION Locomotor activity and rearing & grooming scores of drug treated animals are compared with controls treated with reserpine and vehicle only by analysis of variance. Locomotor activity of drug treated group must be more than reserpene treated control.MPTP model in monkeys: MPTP model in monkeys PURPOSE AND RATIONALE N-MPTP ( N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) It has been shown to cause symptoms of Parkinson’s disease in exposed individuals. When administered to primates(monkeys as in this case) this compound causes a partial destruction of basal ganglia (by ETC blockade) and a syndrome that resembles Parkinson’s disease. 31Circling behavior in nigrostriatal lesioned rats: Circling behavior in nigrostriatal lesioned rats PURPOSE AND RATIONALE Unilateral lesion of the dopaminergic nigrostriatal pathway in the rat by the neurotoxin 6-hydroxydopamine (6OHDA) induces hypersensitivity of the postsynaptic dopaminergic receptors in the striatum of the lesioned side. The rats rotate in a circling chamber in a direction towards the lesioned side ( ipsilateral ) when an indirect acting compound such as amphetamine is administered. but to the opposite direction ( contralateral ) when a direct acting dopamine agonist, e.g., apomorphine , or the dopamine precursor L-dopa is given. Therefore, this test can be used for the study of central dopamine function and the evaluation of dopamine antagonists and agonists, particularly the activity of novel antiparkinsonian drugs. 32Elevated body swing test: Elevated body swing test PURPOSE AND RATIONALE Borlongan and Sanberg (1995) proposed the elevated body swing test as a measure of asymmetrical motor behavior of hemi parkinsonian animals in a drug-free state. The total number of swings made to each side is divided by the overall total number of swings made to both sides to get percentages of left andright swings. The criterion of biased swing is set at 70 % or higher . 6-OHDA-lesioned rats exhibit rightbiased swings of 70% or higher compared to normal rats. 33Skilled paw reaching in rats: Skilled paw reaching in rats PURPOSE AND RATIONALE The term “staircase test” mentioned in this context, has nothing to do with the staircase test for evaluating anxiolytic activity in rats. Unilateral injection of 6-OHDA into the medial forebrain bundle results in an impairment of paw reaching on both sides which can be ameliorated by drug treatment or transplantation of a nigral cell suspension. The number of Saccharinpellets eaten during the test period indicates the rat’s success in grasping and retrieving the pellets; the number of steps from which pellets have been removed provides an index of the attempts to reach the food and how far the rat can reach; the number of missed pellets remaining at the end of the test on the floor of the side compartment indicates a lack of sensorimotor coordination in grasping and retrieving the pellets The apparatus has been developed after earlier studies of motor cortex, nigrostriatal dopamine and caudate- putamen to skilled forelimb use in the rat hich is impairedin PD. 34INVITRO METHODS : INVITRO METHODS EXPERIMENT USING RAT STAITAL SLICES : Striatum in brain is primarily affected in parkinsonism.The release of the neurotransmitter like dopamine and acetylcholine in response to test agent serve as a good in vitro marker of its activity. Male spargue dawley rats(150-250g)are decapitated,the skull is opened. Right and left striata are removed & placed in ice-cold krebs solution. 35PowerPoint Presentation: 36 The striata is cut into 0.4mm thick slices using a tissue chopper. The slices are kept floating for 30 min in krebs solution & gassed with 95% o2 & 5% co2 at room temperature. The slices are labeled by incubating for 30 min at 37C with [3H]dopamine (5 μ ci /ml) &[14c] choline (2 μ ci /ml)in the presence of 0.15mM pargyline chloride & 0.1mM ascorbic acid. Labeled slices are transferred to superfusion chambers & perfused with krebs solutions at 37c at flow rate of 0.5ml/minPowerPoint Presentation: 37 After washing & stabilization 5min fraction of superfusate are collected. The perfusion buffer contains 1 μ M nomifensine to inhibit dopamine reuptake & 10 μ M hemicholinium to inhibit choline uptake. The slices are subjected to field strength to the current strength of 10-15mA/cm2 & pulse duration of 2msec at stimulation frequency of 3Hz for 5min. Drugs to be tested are present in the superfusion fluid.PowerPoint Presentation: 38 The radioactivity in the superfusate samples & in the tissue is determined by liquid scintillation counting. The radiolabelled choline method makes it possible to study Ach release in vitro without inhibiting cholinestrase , thus minimizing auto inhibition of transmitter release caused by accumulation of unhydrolised of Ach.DOPOMINE STUMILATED ADENYLY CYCLASE ACTIVITY: DOPOMINE STUMILATED ADENYLY CYCLASE ACTIVITY Male sprague - dawley (150-250g) are decapitated & right & left striata are removed. Striatal tissue is homogenized by teflon homogenizer in chilled buffer containing 10mM imidazole , 2mM EDTA & 10% sucrose ph 7.3. Homogenate is centrifuged at thousand g for10min & supernatant is recentifuged at 27000g for20min. The pellet obtained is washed twice & suspended in 10mM imidazole , ph 7.3. Membrane protein is determined by bradfords method using bovine serum. 39PowerPoint Presentation: 40 Adenylyl cyclase activity is measured by calculating the conversion rate of (32p) ATP to (32p) cAMP . The assay is perform in 250 μ l solution containing imidazole , mgcl2 papaverine dithiothreitol , ATP,GTP,phspocreatine,creatine phosphokinase . The reaction mixture is preincubated at 30c for 5min, the reaction is initiated by adding membrane proteins and incubated for 10min. The reaction is terminated by adding stopping solution( ATP,SDS,cAMP ). Formed (32p) cAMP is separated from (32p) ATP by chromatography.References :: References : Drug Discovery and Evaluation- H. Gerhard Vogel. Drug screening methods – SK Gupta. Textbook of pathology- harsh mohan . Principlers of anatomy & physiology - tortora & grabowski . Rang & dale. Internet source. 41QUESTION:: QUESTION: Preclinical models for Anti parkinson disease. (5 MARKS). 42PowerPoint Presentation: 43 THE END You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.